PROJECT SUMMARY Current HIV antiretroviral treatment successfully controls viral replication and has transformed HIV-infection from a fatal illness to a manageable chronic condition. However, despite suppression of viral replication during treatment, studies have shown that pools of latent viral reservoirs remain detectable, which fuel viral rebound when antiviral suppression treatment is interrupted. These viral reservoirs are established almost immediately upon infection when HIV irreversibly integrates its viral genome into human DNA. Viral reservoirs are extremely durable, not susceptible to therapeutic effects of currently available antiretroviral agents, and have been refractory to recent experimental treatment approaches. HIV infection is also characterized by a high level of intrahost genotypic diversity of viral quasispecies. In addition to genetic diversity associated base substitution mutations, pools of viral DNA genomes recovered from chronically-infected patients under prolonged suppressive therapy often contain high frequencies of genome-truncated and/or hypermutated, non-replication- competent viral DNA genomes. Only a small fraction of proviral genomes in these patients are genome-intact and may lead to productive viral replication and virologic rebound in the absence of treatment. Furthermore, recent studies by Dr. Lee and other groups have shown clear evidence that HIV-infected cells by both genome- intact and genome-defective proviruses can clonally expand. Such clonal-expansion of infected cells carrying genome-intact HIV proviruses suggests an important mechanism of HIV persistence. However, longitudinal dynamics and mechanism of clonal expansion has not been studied thoroughly, and the diversity between patients not well characterized. Importantly, our current understanding of HIV reservoirs has been derived almost exclusively from studies on a strain called subtype B HIV-1, the predominate viral subtype affecting first- world nations. In contrast, subtype C HIV-1 subtypes is the most prevalent HIV-1 strain globally, accounting approximately 50% of the global HIV-1 burden. In this R21 application, we propose to longitudinally track and compare the genotypic evolution of HIV reservoirs in subtype B and C. We propose to examine clonal expansion of HIV-infected cells. Specifically, we hypothesize that longitudinal examination of HIV DNA genomes and integration sites will reveal features unique to viral species that persist during suppressive therapy, that these features will differ between HIV subtype B versus C, and that these features will be associated with the immunological profile changes in an individual. This study will generate the largest cross-HIV-subtype reservoir database with linked clinical and immunological data. Viral genotypic data will be used to develop subtype- specific assays and bioinformatics pipelines for the study of HIV persistence.